1. Field of the Invention
The present invention relates to novel N-acetyl-.beta.-D-glucosamine derivatives, a process for preparation thereof, a method for determining N-acetyl-.beta.-D-glucosaminidase activity and application to reagents for assaying N-acetyl-.beta.-D-glucosaminidase activity.
2. Description of the Prior Art
N-Acetyl-.beta.-D-glucosaminidase (hereinafter simply referred to as NAGase) is one of enzymes in lysosome distributed in the kidney tubular epithelium in large quantities and participates in decomposition of glycoprotein or mucopolysaccharide. It is recognized that urinary NAGase increases in various renal diseases such as acute renal deficiency, glomerulonephritis, etc. or in postoperative kidney. It is also recognized that NAGase increases in the case of diabetes not only in urine but in serum. As an aid of diagnosis and course observation of various renal diseases and also as an index of studies of a drug on renal toxicity, attention has been brought to assay for NAGase in clinical field and animal experiments.
As substrates for determining NAGase activity hitherto known, there are, for example, p-nitrophenyl-N-acetyl-.beta.-D-glucosaminide [Methods Enzymol., 28, 702 (1972)] and 4-methylumbelliferyl-N-acetyl-.beta.-D-glucosaminide [Clinica. Chemica. Acta., 24, 189 (1969)] and m-cresolsulfonephthaleinyl-N-acetyl-.beta.-D-glucosaminide [Clin. Chem., 29, 1713 (1983)].
In case that p-nitrophenyl-N-acetyl-.beta.-D-glucosaminide described above is used as substrate for determination of NAGase activity, however, the substrate encounters defects that it is affected by yellow substances in urine to increase blank data, since the formed p-nitrophenol is measured at a wavelength at about 400 nm and measurement accuracy is seriously reduced, etc. 4-Methylumbelliferyl-N-acetyl-.beta.-D-glucosaminide involves defects that it is also affected in blank data and requires special devices such as a fluorophotometer, etc. Furthermore, in the case of using p-nitrophenyl-N-acetyl-.beta.-D-glucosaminide and m-cresolsulfonephthaleinyl-N-acetyl-.beta.-D-glucosaminide, aglycone formed by the action of enzyme is colorimetrically determined in such a highly alkaline pH region of the reaction solution as approximately 10 to 11 so that the enzyme reaction must be discontinued; hence, the substrate is disadvantageous in that continuous determination of the enzyme activity is performed only with extreme difficulty.